                                SEQUENCE LISTING

<110> Thanos, Christopher D.
      Murray, Christopher J.
      Yang, Junhao
      Stephenson, Heather
      Sutro Biopharma, Inc.

<120> Proteolytic Inactivation of Select Proteins
  in Bacterial Extracts for Improved Expression
  

<130> 91200-889617

<140> WO Not yet assigned 
<141> Not yet assigned  

<150> US 61/713,245       
<151> 2012-10-12  

<160> 172

<170> FastSEQ for Windows Version 4.0

<210> 1
<211> 360
<212> PRT
<213> Escherichia coli

<220> 
<223> Release Factor 1 (RF1)

<400> 1
Met Lys Pro Ser Ile Val Ala Lys Leu Glu Ala Leu His Glu Arg His
 1               5                  10                  15      
Glu Glu Val Gln Ala Leu Leu Gly Asp Ala Gln Thr Ile Ala Asp Gln
            20                  25                  30          
Glu Arg Phe Arg Ala Leu Ser Arg Glu Tyr Ala Gln Leu Ser Asp Val
        35                  40                  45              
Ser Arg Cys Phe Thr Asp Trp Gln Gln Val Gln Glu Asp Ile Glu Thr
    50                  55                  60                  
Ala Gln Met Met Leu Asp Asp Pro Glu Met Arg Glu Met Ala Gln Asp
65                  70                  75                  80  
Glu Leu Arg Glu Ala Lys Glu Lys Ser Glu Gln Leu Glu Gln Gln Leu
                85                  90                  95      
Gln Val Leu Leu Leu Pro Lys Asp Pro Asp Asp Glu Arg Asn Ala Phe
            100                 105                 110         
Leu Glu Val Arg Ala Gly Thr Gly Gly Asp Glu Ala Ala Leu Phe Ala
        115                 120                 125             
Gly Asp Leu Phe Arg Met Tyr Ser Arg Tyr Ala Glu Ala Arg Arg Trp
    130                 135                 140                 
Arg Val Glu Ile Met Ser Ala Ser Glu Gly Glu His Gly Gly Tyr Lys
145                 150                 155                 160 
Glu Ile Ile Ala Lys Ile Ser Gly Asp Gly Val Tyr Gly Arg Leu Lys
                165                 170                 175     
Phe Glu Ser Gly Gly His Arg Val Gln Arg Val Pro Ala Thr Glu Ser
            180                 185                 190         
Gln Gly Arg Ile His Thr Ser Ala Cys Thr Val Ala Val Met Pro Glu
        195                 200                 205             
Leu Pro Asp Ala Glu Leu Pro Asp Ile Asn Pro Ala Asp Leu Arg Ile
    210                 215                 220                 
Asp Thr Phe Arg Ser Ser Gly Ala Gly Gly Gln His Val Asn Thr Thr
225                 230                 235                 240 
Asp Ser Ala Ile Arg Ile Thr His Leu Pro Thr Gly Ile Val Val Glu
                245                 250                 255     
Cys Gln Asp Glu Arg Ser Gln His Lys Asn Lys Ala Lys Ala Leu Ser
            260                 265                 270         
Val Leu Gly Ala Arg Ile His Ala Ala Glu Met Ala Lys Arg Gln Gln
        275                 280                 285             
Ala Glu Ala Ser Thr Arg Arg Asn Leu Leu Gly Ser Gly Asp Arg Ser
    290                 295                 300                 
Asp Arg Asn Arg Thr Tyr Asn Phe Pro Gln Gly Arg Val Thr Asp His
305                 310                 315                 320 
Arg Ile Asn Leu Thr Leu Tyr Arg Leu Asp Glu Val Met Glu Gly Lys
                325                 330                 335     
Leu Asp Met Leu Ile Glu Pro Ile Ile Gln Glu His Gln Ala Asp Gln
            340                 345                 350         
Leu Ala Ala Leu Ser Glu Gln Glu
        355                 360 


<210> 2
<211> 365
<212> PRT
<213> Escherichia coli

<220> 
<223> Release Factor 2 (RF2)

<400> 2
Met Phe Glu Ile Asn Pro Val Asn Asn Arg Ile Gln Asp Leu Thr Glu
 1               5                  10                  15      
Arg Ser Asp Val Leu Arg Gly Tyr Leu Asp Tyr Asp Ala Lys Lys Glu
            20                  25                  30          
Arg Leu Glu Glu Val Asn Ala Glu Leu Glu Gln Pro Asp Val Trp Asn
        35                  40                  45              
Glu Pro Glu Arg Ala Gln Ala Leu Gly Lys Glu Arg Ser Ser Leu Glu
    50                  55                  60                  
Ala Val Val Asp Thr Leu Asp Gln Met Lys Gln Gly Leu Glu Asp Val
65                  70                  75                  80  
Ser Gly Leu Leu Glu Leu Ala Val Glu Ala Asp Asp Glu Glu Thr Phe
                85                  90                  95      
Asn Glu Ala Val Ala Glu Leu Asp Ala Leu Glu Glu Lys Leu Ala Gln
            100                 105                 110         
Leu Glu Phe Arg Arg Met Phe Ser Gly Glu Tyr Asp Ser Ala Asp Cys
        115                 120                 125             
Tyr Leu Asp Ile Gln Ala Gly Ser Gly Gly Thr Glu Ala Gln Asp Trp
    130                 135                 140                 
Ala Ser Met Leu Glu Arg Met Tyr Leu Arg Trp Ala Glu Ser Arg Gly
145                 150                 155                 160 
Phe Lys Thr Glu Ile Ile Glu Glu Ser Glu Gly Glu Val Ala Gly Ile
                165                 170                 175     
Lys Ser Val Thr Ile Lys Ile Ser Gly Asp Tyr Ala Tyr Gly Trp Leu
            180                 185                 190         
Arg Thr Glu Thr Gly Val His Arg Leu Val Arg Lys Ser Pro Phe Asp
        195                 200                 205             
Ser Gly Gly Arg Arg His Thr Ser Phe Ser Ser Ala Phe Val Tyr Pro
    210                 215                 220                 
Glu Val Asp Asp Asp Ile Asp Ile Glu Ile Asn Pro Ala Asp Leu Arg
225                 230                 235                 240 
Ile Asp Val Tyr Arg Thr Ser Gly Ala Gly Gly Gln His Val Asn Arg
                245                 250                 255     
Thr Glu Ser Ala Val Arg Ile Thr His Ile Pro Thr Gly Ile Val Thr
            260                 265                 270         
Gln Cys Gln Asn Asp Arg Ser Gln His Lys Asn Lys Asp Gln Ala Met
        275                 280                 285             
Lys Gln Met Lys Ala Lys Leu Tyr Glu Leu Glu Met Gln Lys Lys Asn
    290                 295                 300                 
Ala Glu Lys Gln Ala Met Glu Asp Asn Lys Ser Asp Ile Gly Trp Gly
305                 310                 315                 320 
Ser Gln Ile Arg Ser Tyr Val Leu Asp Asp Ser Arg Ile Lys Asp Leu
                325                 330                 335     
Arg Thr Gly Val Glu Thr Arg Asn Thr Gln Ala Val Leu Asp Gly Ser
            340                 345                 350         
Leu Asp Gln Phe Ile Glu Ala Ser Leu Lys Ala Gly Leu
        355                 360                 365 


<210> 3
<211> 317
<212> PRT
<213> Escherichia coli

<220> 
<223> Outer Membrane Protein T1 (OmpT1)

<220> 
<221> SIGNAL         
<222> (1)...(20)
<223> signal peptide

<400> 3
Met Arg Ala Lys Leu Leu Gly Ile Val Leu Thr Thr Pro Ile Ala Ile
-20                 -15                 -10                 -5  
Ser Ser Phe Ala Ser Thr Glu Thr Leu Ser Phe Thr Pro Asp Asn Ile
                 1               5                  10          
Asn Ala Asp Ile Ser Leu Gly Thr Leu Ser Gly Lys Thr Lys Glu Arg
        15                  20                  25              
Val Tyr Leu Ala Glu Glu Gly Gly Arg Lys Val Ser Gln Leu Asp Trp
    30                  35                  40                  
Lys Phe Asn Asn Ala Ala Ile Ile Lys Gly Ala Ile Asn Trp Asp Leu
45                  50                  55                  60  
Met Pro Gln Ile Ser Ile Gly Ala Ala Gly Trp Thr Thr Leu Gly Ser
                65                  70                  75      
Arg Gly Gly Asn Met Val Asp Gln Asp Trp Met Asp Ser Ser Asn Pro
            80                  85                  90          
Gly Thr Trp Thr Asp Glu Ser Arg His Pro Asp Thr Gln Leu Asn Tyr
        95                  100                 105             
Ala Asn Glu Phe Asp Leu Asn Ile Lys Gly Trp Leu Leu Asn Glu Pro
    110                 115                 120                 
Asn Tyr Arg Leu Gly Leu Met Ala Gly Tyr Gln Glu Ser Arg Tyr Ser
125                 130                 135                 140 
Phe Thr Ala Arg Gly Gly Ser Tyr Ile Tyr Ser Ser Glu Glu Gly Phe
                145                 150                 155     
Arg Asp Asp Ile Gly Ser Phe Pro Asn Gly Glu Arg Ala Ile Gly Tyr
            160                 165                 170         
Lys Gln Arg Phe Lys Met Pro Tyr Ile Gly Leu Thr Gly Ser Tyr Arg
        175                 180                 185             
Tyr Glu Asp Phe Glu Leu Gly Gly Thr Phe Lys Tyr Ser Gly Trp Val
    190                 195                 200                 
Glu Ser Ser Asp Asn Asp Glu His Tyr Asp Pro Gly Lys Arg Ile Thr
205                 210                 215                 220 
Tyr Arg Ser Lys Val Lys Asp Gln Asn Tyr Tyr Ser Val Ala Val Asn
                225                 230                 235     
Ala Gly Tyr Tyr Val Thr Pro Asn Ala Lys Val Tyr Val Glu Gly Ala
            240                 245                 250         
Trp Asn Arg Val Thr Asn Lys Lys Gly Asn Thr Ser Leu Tyr Asp His
        255                 260                 265             
Asn Asn Asn Thr Ser Asp Tyr Ser Lys Asn Gly Ala Gly Ile Glu Asn
    270                 275                 280                 
Tyr Asn Phe Ile Thr Thr Ala Gly Leu Lys Tyr Thr Phe
285                 290                 295         


<210> 4
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 4
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 5
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 5
Gln Gln Ala Glu Ala Ser Thr Arg Arg Lys Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 6
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296R mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 6
Gln Gln Ala Glu Ala Ser Thr Arg Arg Arg Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 7
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic L297K mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 7
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Lys Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 8
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic L297R mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 8
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Arg Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 9
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic L297V mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 9
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Val Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 10
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic L298K mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 10
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Leu Lys Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 11
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic L298R mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 11
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Leu Arg Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 12
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K,L297K mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 12
Gln Gln Ala Glu Ala Ser Thr Arg Arg Lys Lys Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 13
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K,L297R = A13 mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 13
Gln Gln Ala Glu Ala Ser Thr Arg Arg Lys Arg Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 14
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K,L297V mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 14
Gln Gln Ala Glu Ala Ser Thr Arg Arg Lys Val Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 15
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K,L297V mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 15
Gln Gln Ala Glu Ala Ser Thr Arg Arg Arg Lys Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 16
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296R,L297R mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 16
Gln Gln Ala Glu Ala Ser Thr Arg Arg Arg Arg Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 17
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296R,L297V mutation in the switch loop region
      of Release Factor 1 (RF1), amino acids 287 to 304

<400> 17
Gln Gln Ala Glu Ala Ser Thr Arg Arg Arg Val Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 18
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K,L297K,L298K mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 18
Gln Gln Ala Glu Ala Ser Thr Arg Arg Lys Lys Lys Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 19
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K,L297K,L298R mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 19
Gln Gln Ala Glu Ala Ser Thr Arg Arg Lys Lys Arg Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 20
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K,L297R,L298K = A17 mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 20
Gln Gln Ala Glu Ala Ser Thr Arg Arg Lys Arg Lys Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 21
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296K,L297R,L298R = A18 mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 21
Gln Gln Ala Glu Ala Ser Thr Arg Arg Lys Arg Arg Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 22
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296R,L297R,L298R mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 22
Gln Gln Ala Glu Ala Ser Thr Arg Arg Arg Arg Arg Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 23
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296R,L297K,L298R mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 23
Gln Gln Ala Glu Ala Ser Thr Arg Arg Arg Lys Arg Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 24
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296R,L297R,L298K mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 24
Gln Gln Ala Glu Ala Ser Thr Arg Arg Arg Arg Lys Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 25
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic N296R,L297K,L298K mutation in the switch loop
      region of Release Factor 1 (RF1), amino acids 287 to 304

<400> 25
Gln Gln Ala Glu Ala Ser Thr Arg Arg Arg Lys Lys Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 26
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 26
Gln Ala Arg Arg Gly Ser Thr Arg Arg Asn Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 27
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 27
Gln Gln Ala Arg Arg Gly Thr Arg Arg Asn Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 28
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 28
Gln Gln Ala Ala Arg Arg Gly Arg Arg Asn Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 29
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 29
Gln Gln Ala Glu Ala Arg Arg Gly Arg Asn Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 30
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 30
Gln Gln Ala Glu Ala Ala Arg Arg Gly Asn Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 31
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 31
Gln Gln Ala Glu Ala Ser Ala Arg Arg Gly Leu Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 32
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 32
Gln Gln Ala Glu Ala Ser Thr Ala Arg Arg Gly Leu Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 33
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 33
Gln Gln Ala Glu Ala Ser Thr Arg Ala Arg Arg Gly Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 34
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 34
Gln Gln Ala Glu Ala Ser Thr Arg Arg Ala Arg Arg Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 35
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 35
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Ala Arg Arg Gly Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 36
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 36
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Leu Ala Arg Arg Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 37
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 37
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Leu Leu Ala Arg Arg Gly
 1               5                  10                  15      
Arg Ser
        

<210> 38
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 38
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Leu Leu Gly Ala Arg Arg
 1               5                  10                  15      
Gly Ser
        

<210> 39
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 39
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Leu Leu Gly Ser Ala Arg
 1               5                  10                  15      
Arg Gly
        

<210> 40
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 40
Gln Gln Ala Glu Ala Ser Thr Arg Arg Asn Leu Leu Gly Ser Gly Ala
 1               5                  10                  15      
Arg Arg
        

<210> 41
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 41
Gln Gln Ala Trp Leu Ala Ala Arg Arg Gly Arg Gly Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 42
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 42
Gln Gln Ala Glu Trp Leu Ala Ala Arg Arg Gly Arg Gly Ser Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 43
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 43
Gln Gln Ala Glu Ala Trp Leu Ala Ala Arg Arg Gly Arg Gly Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 44
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 44
Gln Gln Trp Gly Gly Arg Trp Ala Arg Lys Lys Gly Thr Ile Gly Asp
 1               5                  10                  15      
Arg Ser
        

<210> 45
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 45
Gln Gln Ala Trp Gly Gly Arg Trp Ala Arg Lys Lys Gly Thr Ile Asp
 1               5                  10                  15      
Arg Ser
        

<210> 46
<211> 18
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide sequence inserted
      in the switch loop region of Release Factor 1
      (RF1), amino acids 287 to 304

<400> 46
Gln Gln Ala Glu Trp Gly Gly Arg Trp Ala Arg Lys Lys Gly Thr Ile
 1               5                  10                  15      
Arg Ser
        

<210> 47
<211> 4
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide

<400> 47
Ala Arg Arg Gly
 1              


<210> 48
<211> 9
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide

<400> 48
Trp Leu Ala Ala Arg Arg Gly Arg Gly
 1               5                  


<210> 49
<211> 12
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic OmpT cleavage peptide

<400> 49
Trp Gly Gly Arg Trp Ala Arg Lys Lys Gly Thr Ile
 1               5                  10          


<210> 50
<211> 9
<212> PRT
<213> Artificial Sequence

<220> 
<223> synthetic C-terminal sequence of short form of
      lambda phage Gam protein (GamS)

<400> 50
Gly Gly Ser His His His His His His
 1               5                  


<210> 51
<211> 42
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic short form of lambda phage Gam protein
      (GamS) gene amplification primer

<400> 51
atatatcata tgaacgctta ttacattcag gatcgtcttg ag                    42

<210> 52
<211> 72
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic short form of lambda phage Gam protein
      (GamS) gene amplification primer

<400> 52
atatatgtcg acttaatgat gatgatgatg atgagaaccc cctacctctg aatcaatatc 60
aacctggtgg tg                                                     72

<210> 53
<211> 58
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic 5'-fragment including T7 promoter, constant region
      of the N-terminal sequence and the mutation site first step
      PCR amplification oligo primer 5chiT2PT7

<400> 53
gcgtactagc gtaccacgtg gctggtggcc gattcattaa tgcagctggc acgacagg   58

<210> 54
<211> 60
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic 3'-fragment including mutation site, constant
      C-terminal region and T7 terminator sequences first step PCR
      amplification oligo primer 3chiT2TT7

<400> 54
gcgtactagc gtaccacgtg gctggtggcg gtgagttttc tccttcatta cagaaacggc 60


<210> 55
<211> 28
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic 5'-fragment and 3'-fragment assembly
      overlapping second step PCR single primer 5chiT2

<400> 55
gcgtactagc gtaccacgtg gctggtgg                                    28

<210> 56
<211> 35
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic WT RF1 variant OmpT cleavage site
      screening forward primer

<400> 56
gctcgatgat cctgaaatgc gtgagatggc gcagg                            35

<210> 57
<211> 35
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic M74R RF1 variant OmpT cleavage site
      screening forward primer

<400> 57
gctcgatgat cctgaacgcc gtgagatggc gcagg                            35

<210> 58
<211> 38
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic E76K RF1 variant OmpT cleavage site
      screening forward primer

<400> 58
cgatgatcct gaaatgcgta agatggcgca ggatgaac                         38

<210> 59
<211> 40
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic E84K RF1 variant OmpT cleavage site
      screening forward primer

<400> 59
caggatgaac tgcgcaaagc taaagaaaaa agcgagcaac                       40

<210> 60
<211> 40
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic A85R RF1 variant OmpT cleavage site
      screening forward primer

<400> 60
caggatgaac tgcgcgaacg taaagaaaaa agcgagcaac                       40

<210> 61
<211> 44
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic E87R RF1 variant OmpT cleavage site
      screening forward primer

<400> 61
ggatgaactg cgcgaagcta aacgtaaaag cgagcaactg gaac                  44

<210> 62
<211> 38
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic E108R RF1 variant OmpT cleavage site
      screening forward primer

<400> 62
gccaaaagat cctgatgacc gtcgtaacgc cttcctcg                         38

<210> 63
<211> 34
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic T293R RF1 variant OmpT cleavage site
      screening forward primer

<400> 63
caacaggccg aagcgtctcg ccgtcgtaac ctgc                             34

<210> 64
<211> 34
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K RF1 variant OmpT cleavage site
      screening forward primer

<400> 64
gcgtctaccc gtcgtaaact gctggggagt ggcg                             34

<210> 65
<211> 36
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic S304K RF1 variant OmpT cleavage site
      screening forward primer

<400> 65
gggagtggcg atcgcaagga ccgtaaccgt acttac                           36

<210> 66
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297V RF1 variant OmpT cleavage
      site screening forward primer

<400> 66
cgaagcgtct acccgtcgta aagttctggg gagtggcgat cgcagc                46

<210> 67
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297K RF1 variant OmpT cleavage
      site screening forward primer

<400> 67
cgaagcgtct acccgtcgta aaaagctggg gagtggcgat cgcagc                46

<210> 68
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297R RF1 variant OmpT cleavage
      site screening forward primer

<400> 68
cgaagcgtct acccgtcgta aacgtctggg gagtggcgat cgcagc                46

<210> 69
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296R/L297V RF1 variant OmpT cleavage
      site screening forward primer

<400> 69
cgaagcgtct acccgtcgtc gcgttctggg gagtggcgat cgcagc                46

<210> 70
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296R/L297K RF1 variant OmpT cleavage
      site screening forward primer

<400> 70
cgaagcgtct acccgtcgtc gcaagctggg gagtggcgat cgcagc                46

<210> 71
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296R/L297R RF1 variant OmpT cleavage
      site screening forward primer

<400> 71
cgaagcgtct acccgtcgtc gccgtctggg gagtggcgat cgcagc                46

<210> 72
<211> 55
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297R/L298K RF1 variant OmpT
      cleavage site screening forward primer

<400> 72
caggccgaag cgtctacccg tcgtaaacgt aaggggagtg gcgatcgcag cgacc      55

<210> 73
<211> 55
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297R/L298R RF1 variant OmpT
      cleavage site screening forward primer

<400> 73
caggccgaag cgtctacccg tcgtaaacgt cgcgggagtg gcgatcgcag cgacc      55

<210> 74
<211> 35
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic WT RF1 variant OmpT cleavage site
      screening reverse primer

<400> 74
cctgcgccat ctcacgcatt tcaggatcat cgagc                            35

<210> 75
<211> 35
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic M74R RF1 variant OmpT cleavage site
      screening reverse primer

<400> 75
cctgcgccat ctcacggcgt tcaggatcat cgagc                            35

<210> 76
<211> 38
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic E76K RF1 variant OmpT cleavage site
      screening reverse primer

<400> 76
gttcatcctg cgccatctta cgcatttcag gatcatcg                         38

<210> 77
<211> 40
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic E84K RF1 variant OmpT cleavage site
      screening reverse primer

<400> 77
gttgctcgct tttttcttta gctttgcgca gttcatcctg                       40

<210> 78
<211> 40
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic A85R RF1 variant OmpT cleavage site
      screening reverse primer

<400> 78
gttgctcgct tttttcttta cgttcgcgca gttcatcctg                       40

<210> 79
<211> 44
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic E87R RF1 variant OmpT cleavage site
      screening reverse primer

<400> 79
gttccagttg ctcgctttta cgtttagctt cgcgcagttc atcc                  44

<210> 80
<211> 38
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic E108R RF1 variant OmpT cleavage site
      screening reverse primer

<400> 80
cgaggaaggc gttacgacgg tcatcaggat cttttggc                         38

<210> 81
<211> 34
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic T293R RF1 variant OmpT cleavage site
      screening reverse primer

<400> 81
gcaggttacg acggcgagac gcttcggcct gttg                             34

<210> 82
<211> 34
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K RF1 variant OmpT cleavage site
      screening reverse primer

<400> 82
cgccactccc cagcagttta cgacgggtag acgc                             34

<210> 83
<211> 36
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic S304K RF1 variant OmpT cleavage site
      screening reverse primer

<400> 83
gtaagtacgg ttacggtcct tgcgatcgcc actccc                           36

<210> 84
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297V RF1 variant OmpT cleavage
      site screening reverse primer

<400> 84
gctgcgatcg ccactcccca gaactttacg acgggtagac gcttcg                46

<210> 85
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297K RF1 variant OmpT cleavage
      site screening reverse primer

<400> 85
gctgcgatcg ccactcccca gctttttacg acgggtagac gcttcg                46

<210> 86
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297R RF1 variant OmpT cleavage
      site screening reverse primer

<400> 86
gctgcgatcg ccactcccca gacgtttacg acgggtagac gcttcg                46

<210> 87
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296R/L297V RF1 variant OmpT cleavage
      site screening reverse primer

<400> 87
gctgcgatcg ccactcccca gaacgcgacg acgggtagac gcttcg                46

<210> 88
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296R/L297K RF1 variant OmpT cleavage
      site screening reverse primer

<400> 88
gctgcgatcg ccactcccca gcttgcgacg acgggtagac gcttcg                46

<210> 89
<211> 46
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296R/L297R RF1 variant OmpT cleavage
      site screening reverse primer

<400> 89
gctgcgatcg ccactcccca gacggcgacg acgggtagac gcttcg                46

<210> 90
<211> 55
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297R/L298K RF1 variant OmpT
      cleavage site screening reverse primer

<400> 90
ggtcgctgcg atcgccactc cccttacgtt tacgacgggt agacgcttcg gcctg      55

<210> 91
<211> 55
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic N296K/L297R/L298R RF1 variant OmpT
      cleavage site screening reverse primer

<400> 91
ggtcgctgcg atcgccactc ccgcgacgtt tacgacgggt agacgcttcg gcctg      55

<210> 92
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 0 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 92
gccgaagcgt ctacccgtcg taacctgctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 93
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 1 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 93
cgccgtggtt ctacccgtcg taacctgctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 94
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 2 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 94
gcacgccgtg gtacccgtcg taacctgctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 95
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 3 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 95
gccgcacgcc gtggtcgtcg taacctgctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 96
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 4 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 96
gccgaagcac gccgtggtcg taacctgctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 97
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 5 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 97
gccgaagcgg cacgccgtgg taacctgctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 98
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 6 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 98
gccgaagcgt ctgcacgccg tggtctgctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 99
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 7 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 99
gccgaagcgt ctaccgcacg ccgtggtctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 100
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 8 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 100
gccgaagcgt ctacccgtgc acgccgtggt gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 101
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 9 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 101
gccgaagcgt ctacccgtcg tgcacgccgt ggtagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 102
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 10 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 102
gccgaagcgt ctacccgtcg taacgcacgc cgtggtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 103
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 11 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 103
gccgaagcgt ctacccgtcg taacctggca cgccgtggtg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 104
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 12 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 104
gccgaagcgt ctacccgtcg taacctgctg gcacgccgtg gtcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 105
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 13 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 105
gccgaagcgt ctacccgtcg taacctgctg ggggcacgcc gtggtagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 106
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 14 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 106
gccgaagcgt ctacccgtcg taacctgctg gggagtgcac gccgtggtga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 107
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 15 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 107
gccgaagcgt ctacccgtcg taacctgctg gggagtggcg cacgccgtga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 108
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 16 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 108
gccgaagcgt ctacccgtcg tcgtctgctg gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 109
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 17 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 109
gcctggctgg cagcgcgtcg cggtcgtggc gggagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 110
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 18 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 110
gccgaatggc tggcagcgcg tcgcggtcgt ggcagtggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 111
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 19 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 111
gccgaagcgt ggctggcagc gcgtcgcggt cgtggcggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 112
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 20 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 112
tggggtggcc gttgggctcg caagaaaggt actattggcg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 113
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 21 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 113
gcctggggtg gccgttgggc tcgcaagaaa ggtactattg atcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 114
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 22 RF1 variant OmpT cleavage peptide
      insertion forward oligo primer

<400> 114
gccgaatggg gtggccgttg ggctcgcaag aaaggtacta ttcgcagcga ccgtaaccgt 60
acttacaact tcccg                                                  75

<210> 115
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 0 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 115
gctgcgatcg ccactcccca gcaggttacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 116
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 1 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 116
gctgcgatcg ccactcccca gcaggttacg acgggtagaa ccacggcgtg cttggcgttt 60
tgccatttca gcagc                                                  75

<210> 117
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 2 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 117
gctgcgatcg ccactcccca gcaggttacg acgggtacca cggcgtgcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 118
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 3 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 118
gctgcgatcg ccactcccca gcaggttacg acgaccacgg cgtgcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 119
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 4 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 119
gctgcgatcg ccactcccca gcaggttacg accacggcgt gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 120
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 5 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 120
gctgcgatcg ccactcccca gcaggttacc acggcgtgcc gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 121
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 6 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 121
gctgcgatcg ccactcccca gcagaccacg gcgtgcagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 122
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 7 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 122
gctgcgatcg ccactcccca gaccacggcg tgcggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 123
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 8 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 123
gctgcgatcg ccactcccac cacggcgtgc acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 124
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 9 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 124
gctgcgatcg ccactaccac ggcgtgcacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 125
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 10 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 125
gctgcgatcg ccaccacggc gtgcgttacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 126
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 11 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 126
gctgcgatca ccacggcgtg ccaggttacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 127
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 12 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 127
gctgcgacca cggcgtgcca gcaggttacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 128
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 13 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 128
gctaccacgg cgtgccccca gcaggttacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 129
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 14 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 129
accacggcgt gcactcccca gcaggttacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 130
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 15 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 130
acggcgtgcg ccactcccca gcaggttacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 131
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 16 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 131
gctgcgatcg ccactcccca gcagacgacg acgggtagac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 132
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 17 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 132
gctgcgatcg ccactcccgc cacgaccgcg acgcgctgcc agccaggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 133
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 18 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 133
gctgcgatcg ccactgccac gaccgcgacg cgctgccagc cattcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 134
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 19 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 134
gctgcgatcg ccgccacgac cgcgacgcgc tgccagccac gcttcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 135
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 20 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 135
gctgcgatcg ccaatagtac ctttcttgcg agcccaacgg ccaccccact gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 136
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 21 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 136
gctgcgatca atagtacctt tcttgcgagc ccaacggcca ccccaggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 137
<211> 75
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic No. 22 RF1 variant OmpT cleavage peptide
      insertion reverse oligo primer

<400> 137
gctgcgaata gtacctttct tgcgagccca acggccaccc cattcggcct gttggcgttt 60
tgccatttca gcagc                                                  75

<210> 138
<211> 66
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic RF1 amplification primer 5His-RF1

<400> 138
catatgcatc accatcacca tcacggtggt ggctctaagc cttctatcgt tgccaaactg 60
gaagcc                                                            66

<210> 139
<211> 33
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic RF1 amplification primer 3RF1

<400> 139
gtcgacttat tcctgctcgg acaacgccgc cag                              33

<210> 140
<211> 90
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic Oligonucleotide-Mediated Allelic
      Replacement (OMAR) PCR oligo 1opRF1 KR

<400> 140
gggaagttgt aagtacggtt acggtcgctg cgatcccctg aaccaagacg ctttcgacgg 60
gtagacgctt cggcctgttg gcgttttgcc                                  90

<210> 141
<211> 90
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic Oligonucleotide-Mediated Allelic
      Replacement (OMAR) PCR oligo 1opRF1 KRR

<400> 141
gggaagttgt aagtacggtt acggtcgctg cgatcccctg aaccacgacg ctttcgacgg 60
gtagacgctt cggcctgttg gcgttttgcc                                  90

<210> 142
<211> 90
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic Oligonucleotide-Mediated Allelic
      Replacement (OMAR) PCR oligo 1opRF1 KRK

<400> 142
gggaagttgt aagtacggtt acggtcgctg cgatcccctg aacccttacg ctttcgacgg 60
gtagacgctt cggcctgttg gcgttttgcc                                  90

<210> 143
<211> 22
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic Mismatch Amplification Mutation Assay
      (MAMA) PCR oligo 3KR op-PCR

<400> 143
gcgatcccct gaaccaagac gc                                          22

<210> 144
<211> 21
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic Mismatch Amplification Mutation Assay
      (MAMA) PCR oligo 3 KRR op-PCR

<400> 144
cgatcccctg aaccacgacg c                                           21

<210> 145
<211> 23
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic Mismatch Amplification Mutation Assay
      (MAMA) PCR oligo 3KRK op-PCR

<400> 145
tgcgatcccc tgaaccctta cgc                                         23

<210> 146
<211> 22
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic Mismatch Amplification Mutation Assay
      (MAMA) PCR oligo 5 RF1 op-PCR

<400> 146
cgtgacgggg ataacgaacg cc                                          22

<210> 147
<211> 19
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic rolling circle amplification and primer
      extension sequencing primer T7

<400> 147
taatacgact cactatagg                                              19

<210> 148
<211> 18
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic rolling circle amplification and primer
      extension sequencing primer T7 term

<400> 148
gctagttatt gctcagcg                                               18

<210> 149
<211> 39
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00067_V422 sense oligo primer for introducing
      amber codon in Herceptin

<400> 149
cgttggcagc agggtaatta gttcagctgc agcgttatg                        39

<210> 150
<211> 39
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00067_V422 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 150
cataacgctg cagctgaact aattaccctg ctgccaacg                        39

<210> 151
<211> 42
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00127_S415 sense oligo primer for introducing
      amber codon in Herceptin

<400> 151
gcaagctgac cgtcgataaa tagcgttggc agcagggtaa tg                    42

<210> 152
<211> 42
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00127_S415 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 152
cattaccctg ctgccaacgc tatttatcga cggtcagctt gc                    42

<210> 153
<211> 36
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00128_Q418 sense oligo primer for introducing
      amber codon in Herceptin

<400> 153
cgataaaagc cgttggtagc agggtaatgt gttcag                           36

<210> 154
<211> 36
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00128_Q418 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 154
ctgaacacat taccctgcta ccaacggctt ttatcg                           36

<210> 155
<211> 35
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00114_P343 sense oligo primer for introducing
      amber codon in Herceptin

<400> 155
gcaaagcgaa aggccaatag cgtgaaccgc aggtc                            35

<210> 156
<211> 35
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00114_P343 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 156
gacctgcggt tcacgctatt ggcctttcgc tttgc                            35

<210> 157
<211> 40
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00112_G341 sense oligo primer for introducing
      amber codon in Herceptin

<400> 157
gacgatcagc aaagcgaaat agcaaccgcg tgaaccgcag                       40

<210> 158
<211> 40
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00112_G341 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 158
ctgcggttca cgcggttgct atttcgcttt gctgatcgtc                       40

<210> 159
<211> 41
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00102_K320 sense oligo primer for introducing
      amber codon in Herceptin

<400> 159
gctgaatggt aaagaatact agtgcaaagt gagcaacaag g                     41

<210> 160
<211> 41
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00102_K320 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 160
ccttgttgct cactttgcac tagtattctt taccattcag c                     41

<210> 161
<211> 39
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00066_F404 sense oligo primer for introducing
      amber codon in Herceptin

<400> 161
ctggacagcg acggtagcta gtttctgtat agcaagctg                        39

<210> 162
<211> 39
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00066_F404 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 162
cagcttgcta tacagaaact agctaccgtc gctgtccag                        39

<210> 163
<211> 32
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00113_Q342 sense oligo primer for introducing
      amber codon in Herceptin

<400> 163
gcaaagcgaa aggctagccg cgtgaaccgc ag                               32

<210> 164
<211> 32
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00113_Q342 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 164
ctgcggttca cgcggctagc ctttcgcttt gc                               32

<210> 165
<211> 42
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00096_T299 sense oligo primer for introducing
      amber codon in Herceptin

<400> 165
gtgaggaaca atacaatagc tagtatcgcg tagtgagcgt gc                    42

<210> 166
<211> 42
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00096_T299 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 166
gcacgctcac tacgcgatac tagctattgt attgttcctc ac                    42

<210> 167
<211> 30
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00120_Y373 sense oligo primer for introducing
      amber codon in Herceptin

<400> 167
ggtgaagggc ttttagccga gcgacatcgc                                  30

<210> 168
<211> 30
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00120_Y373 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 168
gcgatgtcgc tcggctaaaa gcccttcacc                                  30

<210> 169
<211> 38
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00094_N297 sense oligo primer for introducing
      amber codon in Herceptin

<400> 169
cgcgtgagga acaatactag agcacgtatc gcgtagtg                         38

<210> 170
<211> 38
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00094_N297 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 170
cactacgcga tacgtgctct agtattgttc ctcacgcg                         38

<210> 171
<211> 38
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00125_F405 sense oligo primer for introducing
      amber codon in Herceptin

<400> 171
gacagcgacg gtagcttcta gctgtatagc aagctgac                         38

<210> 172
<211> 38
<212> DNA
<213> Artificial Sequence

<220> 
<223> synthetic site directed mutagenesis variant
      SP-00125_F405 antisense oligo primer for
      introducing amber codon in Herceptin

<400> 172
gtcagcttgc tatacagcta gaagctaccg tcgctgtc                         38


